Research ArticleEnhanced Crystallinity and Antibacterial of PHBV ScaffoldsIncorporated with Zinc Oxide

Cijun Shuai ,1,2,3 Chen Wang,1 Fangwei Qi ,1 Shuping Peng ,4,5 Wenjing Yang,1

Chongxian He,1 Guoyong Wang,1 and Guowen Qian1

1Institute of Bioadditive Manufacturing, Jiangxi University of Science and Technology, Nanchang 330013, China2State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, China3Shenzhen Institute of Information Technology, Shenzhen 518172, China4NHC Key Laboratory of Carcinogenesis, School of basic Medical Science, Central South University, Changsha, Hunan 410013, China5School of Energy and Machinery Engineering, Jiangxi University of Science and Technology, Nanchang 330013, China

Correspondence should be addressed to Fangwei Qi; qfw@jxust.edu.cn and Shuping Peng; shuping@csu.edu.cn

Received 29 May 2020; Accepted 29 June 2020; Published 8 July 2020

Academic Editor: Xiaoming Li

Copyright © 2020 Cijun Shuai et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has a great potential in bone repair, but unfortunately, the poormechanical properties limit its further application. In this work, zinc oxide (ZnO) nanoparticles were incorporated into PHBVporous scaffold fabricated by selective laser sintering technique. It was because ZnO nanoparticles could provide nucleating sitesfor the orderly stacking of polymer chains, thereby enhancing the crystallinity of PHBV. It was well known that the mechanicalproperties of PHBV scaffold could be enhanced with the increase of crystallinity. More significantly, the released Zn2+ wouldcombine negatively charged cell membranes of bacterial through electrostatic interaction and consequently destructed theprotein structure and resulted in the death of bacterial, which was highly desired in reducing the risk of implant infection.Results indicated that the relative crystallinity of scaffold with 3wt.% ZnO increased remarkably from 38% to 64% compared topure PHBV scaffold, which effectively enhanced the compression strength and modulus by 56% and 51.5%, respectively.Moreover, the scaffold had a favorable antibacterial activity. Cell culture experiments proved that the scaffold could promote thecell behaviors. The positive results demonstrated the scaffold may serve as a potential replacement in bone repair.

1. Introduction

The large bone defects resulted from trauma, infection, andtumor are commonly difficult to heal by their own ability,which seriously affects the patients’ living quality and healthlevel [1]. Artificial bone scaffolds, which enable to provide amechanical and physiological support to cells for in vitrotissue regeneration and/or in vivo implantation, are consideredto be a promising alternative in the treatment of large bonedefects [2, 3]. Among various biomaterials, poly(3-hydroxybu-tyrate-co-3-hydroxyvalerate) (PHBV) has received consider-able attention because of its favorable biocompatibility andbiodegradability. It can degrade in vivo to hydroxybutyric,which is a normal component of human blood [4]. However,

insufficient mechanical performance has limited its furtherapplication in bone repair.

It is well established that themechanical property of PHBVis positively related to its crystallinity [5]. Introducing nanofil-lers is considered to be an effective countermeasure to improvethe crystallinity [6]. For example, Jun et al. [7] have found thatthe crystallinity of PHBV was greatly improved with theincorporation of carbon nanotubes. Kouhi et al. [8] fabricatedPHBV/hydroxyapatite/bredigite scaffolds and found that theincorporation of nanoparticles increased the crystallinity ofPHBVmatrix and subsequently enhanced the Young’s modu-lus and ultimate strength. Zhang et al. [9] prepared PHBV/cel-lulose nanocrystals/silver nanocomposites by using solutioncasting. It was found that the composites showed higher

HindawiJournal of NanomaterialsVolume 2020, Article ID 6014816, 12 pageshttps://doi.org/10.1155/2020/6014816

crystallinity and smaller average crystallite size compared toPHBV. Meanwhile, the tensile strength and modulus of thecomposites were greatly increased by 97.6% and 231%,respectively.

Among various nanofillers, zinc oxide (ZnO) nanoparti-cles possess good biocompatibility, which has been approvedas a biomedical material by Food and Drug Administration(FDA) [10]. More interestingly, ZnO has a favorable antibac-terial ability against a wide a range of bacteria. It can releasezinc ions (Zn2+) to combine with the negatively charged cellmembranes of bacterial through electrostatic interaction andconsequently destructed the protein structure and resulted inthe death of bacterial, which was highly desired in reducingthe risk of implant infection [11]. Furthermore, it can alsogenerate reactive oxygen species (ROS) under the irradiationof ultraviolet light to damage the cell membranes of bacterial[12, 13]. Moreover, Zn is an important trace element in thehuman body and participates in various metabolic pro-cesses [14]. It has been reported that an appropriateamount of Zn can promote bone formation while inhibitingbone resorption.

Although different fabrication methods including solventcasting, particle-leaching, and foam replication have beendeveloped to manufacture bone scaffolds with desirableproperties, these techniques have great difficulty in fabricat-ing bone scaffolds with controllable porous structure andpersonalized shape [15]. It is well established that the porousstructure of the scaffold is conducive to the growth of cells,the transmission of nutrients, and the discharge of metabo-lites, whereas the personalized shape design can meet therequirements of various patients [16, 17]. As an emergingadditive manufacturing technique, selective laser sintering(SLS) can precisely construct bone scaffold with complicatedstructure and customized shape from 3D digital data bysequentially fusing region in a powder bed, layer-by-layer,via a computer-controlled scanning laser beam. More partic-ularly, any powdered biomaterial that will fuse but notdecompose under the irradiation of laser beam can be usedto fabricate scaffold [18].

In this work, PHBV/ZnO scaffolds were manufactured byselective laser sintering. The crystallization behaviors,mechanical performances, and antibacterial activities weresystematically investigated. The relationship between thecrystallization behavior and mechanical properties wasdiscussed in depth. Moreover, in vitro cell behaviors werealso explored. This work attempts to provide an effectivecountermeasure to simultaneously improve the mechanicalproperty and antibacterial activity of PHBV scaffold and alsosupplies a strategy to overcome challenges in rapidly fabricat-ing scaffolds with controllable pore structure.

2. Experiment

2.1. Materials. PHBV powders (molecular weight: 280 kDa,density: 1.25g/cm3) were provided by TianAn BiologicalMaterials Co., Ltd. (Ningbo, China). ZnO nanoparticles withaverage size of 50nm and specific surface area of 15-25m2/gwere supplied by Aladdin Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of PHBV/ZnO Scaffold. The fabricationprocess of three-dimensional porous PHBV/ZnO compositescaffolds is illustrated in Figure 1. In detail, a certain amountof PHBV and ZnO powders was weighted according to thedesigned mass ratio and then poured into a beaker contain-ing ethanol solution. Subsequently, the mixed suspensionswere subjected to mechanical agitation and ultrasonic disper-sion simultaneously for 2 h. Afterwards, the well-mixedsuspensions were filtered and dried in the vacuum dryingoven. Ultimately, the mixed powders could be obtained byusing ball milling.

The scaffolds were manufactured by a laser 3D moldingsystem with independent intellectual property rights. Itmainly consisted of a continuous wave CO2 laser with thewavelength of 10.6μm. The whole processing parameters inthis study were set as follows: laser power 5W, scanningspeed 100mm/s, and scanning space 0.15mm. The three-dimensional porous scaffolds could be obtained throughlayer-by-layer construction. For the convenience of descrip-tion, PHBV scaffold with various ZnO contents of 1, 3, and5wt.%, which were named as PHBV/1nZnO, PHBV/3nZnO,and PHBV/5nZnO, respectively.

2.3. Microstructure and Mechanical Property. The morphol-ogies of the samples were characterized by scanning electronmicroscopy (SEM, Zeiss, Germany). The functional groupsof the scaffolds were analyzed by the Fourier transforminfrared spectroscopy (FTIR, Tianjin Gang Dong Technol-ogy Co. Ltd., China). The phase structures of the scaffoldswere measured by utilizing X-ray diffractometer (XRD,Karlsruhe, Germany). The melting and crystallization perfor-mance of the scaffolds under a constant heating and coolingrate of 10°C/min was measured by differential scanningcalorimeter (DSC, TA, USA). The samples were firstly heatedfrom 30 to 210°C to remove the thermal story of the PHBVand then cooled to 30°C. Afterwards, the samples were heatedto 210°C again. The thermal stabilities of the samples wereinvestigated by thermogravimetric analyzer (TG, PerkinEl-mer, USA). The Zn2+ concentration released by scaffold inthe deionized water was quantitative analyzed via inductivelycoupled plasma optical emission spectrometer (ICP-OES,SPECTROBLUE SOP, Germany). The compression proper-ties of the scaffolds were measured by mechanical testingmachine (CMTS5205, MTS, USA) under a deformation rateof 0.5mm/s.

2.4. Antibacterial Activity. Before the experiment, all experi-mental apparatus and the samples were washed with ethylalcohol under the ultrasound bath and subsequentlysterilized with ultraviolet (UV) for 1 h. The phosphate buffersolution (PBS) was used to dilute the bacterial suspensionsand then seeded to lysogeny broth (LB) culture medium.

The Escherichia coli (E. coli, ATCC 25922) were selectedto explore the antibacterial properties of the scaffolds. Theantibacterial properties of the scaffolds against E. coli werequantitatively evaluated by the bacterial inhibition rate. Indetail, the scaffolds with various ZnO contents wereimmersed in the Petri dish containing bacterial suspensionswith a density of 1 × 106 CFU/mL and cultured for 1, 4, and

2 Journal of Nanomaterials

7 days at 37°C. Then, the optical density of bacterial was alsomeasured by a microplate reader (Beckman, USA) at600nm. The antibacterial rate was calculated as the followingequation:

Antibacterial rate %ð Þ = A2 − A1A1

× 100% ð1Þ

where A2 and A1 were the optical density of the bacterial sus-pensions contained with and without scaffolds, respectively.Each sample was tested for three times.

The morphologies of bacterial on the scaffolds wereobserved by SEM. In detail, the bacterial-scaffold complexeswere taken out from the Petri dish and cleaned with PBS.Afterwards, the bacterial-scaffold complexes were fixed withglutaraldehyde and dehydrated with a series of alcohol.Subsequently, the complex was dried in vacuum drying ovenand finally observed by SEM.

2.5. Cell Response. The cell response of the scaffolds wasevaluated by using human osteoblast-like MG-63 cells. Priorto testing, the cells were cultured in glucose DMEM contain-ing 10% fetal bovine serum at 37°C under a humidifiedenvironment with 5% CO2, and the culture medium wasrefreshed every two days. Before cell seeding, the scaffoldswere washed with PBS and sterilized with UV for 30minfollowed by transferring them into a 12-well dish. Subse-quently, the cells were seeded on scaffolds with a density of1 × 104 cell/mL.

The cell adhesion on scaffold cultured for various periodswas observed by SEM. After 1, 3, and 5 days of cultivation,the cell-scaffold complex was taken out and then washed withPBS. Hereafter, the cells on scaffolds were fixed with glutaral-dehyde and dehydrated with gradient ethanol. Ultimately,the morphologies of the cells on the scaffolds were observed.

Fluorescence microscope (Olympus Co. Ltd., Tokyo,Japan) was adopted to observe the cell proliferation. Thecell-scaffold complex was taken out from medium and

washed with PBS for three times after culturing for 1, 3,and 5 days. Then, the cell-scaffold complex was stained withlive/dead staining agent (PBS solution with 2μM calcein AMand 4μM EthD-1) and continuously incubated for another30min. Finally, fluorescence microscope was selected toobserve the living/dead cell morphologies.

The proliferation of MG-63 cell on scaffolds was quanti-tatively analyzed by the Cell Counting Kit-8 (CCK-8) assay.The cell-scaffold complex was gently washed with PBS andtransferred into a new medium containing CCK-8 reagent(Dojindo Laboratory, Kumamoto, Japan) after 1, 3, and 5days of cultivation. 100μL of medium was moved to a 96-well plate after culture for another 2 hours, and the opticaldensity was measured via a microplate reader (Beckman,USA) at 450nm.

Alkaline phosphatase (ALP), as a specific marker forearly osteoblast differentiation, was observed by ALP stain-ing. After 1, 3, and 5 days of cultivation, the cell-scaffoldcomplex was gently rinsed with PBS and fixed with 4% para-formaldehyde for 20min. Subsequently, ALP staining wascarried out using the ALP staining kit (Wako, Osaka, Japan),and images were photographed via a microscopy.

2.6. Statistical Analysis. One-way analysis of variance(ANOVA) was selected to evaluate the statistical significance.All data were presented as mean ± standard deviations. Thedifference with ∗p < 0:05 was recognized to be significant.

3. Results and Discussion

3.1. Microstructure. The digital photos and the microstruc-tures of the represent scaffold with height of 10mm anddiameter of 10mm are shown in Figure 2(a). Obviously, thescaffold displayed a well porous structure, which was consis-tent with the as-designed models. The pore size of the scaf-fold was approximately 500μm. It had been reported thatthe minimum pore size of the bone scaffold should not be lessthan 100μm to ensure the nutrients transport and cells

Drying

Ball millingSelective laser sinteringScaffold

ZnO

PHBV

Filtering

LaserScanner

Roller

Powderdelivery

Work platform

Powder bed

Sonicating & stirring

50°C

Figure 1: The manufacturing process of the scaffolds.

3Journal of Nanomaterials

growth [19]. Meanwhile, the mechanical performance of thescaffold would be deteriorated with the continuous ascent ofthe pore size, and the maximum pore size should not behigher than 1000μm [20]. It could be concluded that the poresize of the as-prepared scaffold was in the range of100~1000μm and fulfilled the requirement of the bone scaf-fold. As reported in the literature, the dispersion state of thenanofillers in the polymer matrix had a significant influenceon their comprehensive properties [21].

The cryofracture morphologies of the scaffolds aredisplayed in Figure 2(b). It could be obviously found thatthe composite scaffold presented a random and uniformnanofiller distribution with the content of ZnO lower than3wt.%. However, the composite scaffold with 5wt.% ZnOpresented poor nanofiller distribution, with small agglomer-ation composed of a few particles.

3.2. Thermal Behaviors. The thermal stabilities of thescaffold were measured by TG, and the results are shownin Figure 3(a). It could be seen from TG curves that allscaffolds exhibited a single decomposition stage. The initialdecomposition temperature of PHBV scaffolds remarkablyincreased with the increase of ZnO. For instance, the initialdecomposition temperature of PHBV/5nZnO scaffoldincreased from 261.2°C to 288.7°C in comparison with thatof the PHBV scaffold. This improvement might be attrib-uted to the interaction between the hydroxyl group ofZnO and the carbonyl of PHBV, which could form a barriereffect [22]. This barrier effect could prevent the transmissionof the decomposition products. Furthermore, ZnO withexcellent thermal conductivity could accelerate the heatdissipation in the composite and thereby enhance the ther-mal stability of the composite [23, 24]. It could also befound that the char residue of PHBV scaffold was graduallyincreased with increasing ZnO content, which was attrib-uted to the relatively higher decomposition temperature ofZnO nanoparticles [25].

The melting and crystallization behaviors of the scaffoldsare displayed in Figures 3(b) and 3(c). It could be seen fromthe melting curves that pure PHBV scaffold presented twoobvious endothermic peaks located at 160.9°C and 170.6°C,which were due to the melting of the initial crystals and therecrystallized crystals during the DSC heating process,respectively [26]. The endothermic peaks of the compositescaffolds decreased with the increase of ZnO. The crystalliza-tion peak temperature of PHBV scaffolds gradually shifted toa higher temperature, which demonstrated that the nanofil-lers could efficiently promote the crystallization rate of thepolymer (Figure 3(c)) [27]. In addition, the crystallizationpeak became more sharpened in the composite scaffolds,indicating that the nanofillers could efficiently acceleratethe crystallization process of the polymer [28]. The relativecrystallinity of the scaffolds could be calculated by the follow-ing equation:

Xc =ΔHm

ΔH°m ×w× 100%Xc =

ΔHmΔH100 ×w

× 100%: ð2Þ

where ΔHm is the melting enthalpy of PHBV, w is the massfraction of PHBV in the composites, and ΔHο

m is the theoret-ical enthalpy of PHBV (109 J/g) [29]. It could be found thatthere was a remarkable increase in the relative crystallinityof PHBV/3nZnO composite scaffold from 38% to 64% incomparison with that of PHBV scaffolds, which might beattributed to the accelerated nucleation effect of ZnO nano-particles. However, relative crystallinity of the compositescaffolds was decreased with the further increase of nanofil-lers. This might be due to the aggregation of the nanofillers,which hindered the mobility of the polymer chains duringthe crystallization process.

The XRD patterns of the scaffolds are shown inFigure 3(d). There was two obvious peaks located at 2θ of13.4° and 16.9°, being ascribed to the (020) and (110) crystal-line planes of PHBV, respectively [30]. The characteristic

500 𝜇m

40 50 60 70

(a)

bPHBV PHBV/1nZnO

PHBV/3nZnO PHBV/5nZnO

5 𝜇m

5 𝜇m 5 𝜇m

5 𝜇m

(b)

Figure 2: (a) The digital photos and the microstructures of the represent scaffold; (b) the cryofractured morphologies of PHBV andPHBV/nZnO scaffolds.

4 Journal of Nanomaterials

peaks located at 34.5° and 36.1° were ascribed to the (002)and (101) crystalline planes of ZnO, respectively [31].Compared with PHBV, the peak in the composite scaffoldbecame narrower and more intense, which indicated thatthe nanofiller could efficiently promote the crystallization ofPHBV, thereby resulting in the formation of smaller crystals.Moreover, there was no peak shift in the XRD patterns,which revealed that the SLS process did not destroy thecrystal structures of the materials.

The functional groups of the scaffolds were characterizedby FTIR, and the results are shown in Figure 3(e). PurePHBV presented a strong peak situated at 1727 cm−1, whichwas assigned to the telescopic vibration of carbonyl group

[32]. The peaks situated at 1445 and 1300 cm-1, which wereascribed to the asymmetric bending vibration of CH3 andthe in-plane bending vibration of H-C-O [33]. Once ZnOnanoparticles were incorporated, a broad band appeared at3428 cm−1, being attributed to the stretching vibration ofhydroxyl group on the nanoparticle surface [34]. It shouldbe noted that the carbonyl band in the composite scaffoldsbecame more intense and slightly shifted to lower wavenum-ber in comparison with that of pure PHBV, implying thatthere was a hydrogen bond interactions between the carbonylgroup of PHBV and hydroxyl group of the nanoparticles [35].

The wettability of the scaffold had a significant influenceon cell behaviors. It was established that the PHBV presented

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PHBV

𝜃=88.33°

𝜃=58.84°𝜃=63.11°

𝜃=97.06°

𝜃

(f)

Figure 3: (a) TG curves, (b) DSC heating curves, (c) DSC cooling curves, (d) XRD patterns, (e) FTIR spectra, and (f) water contact angles ofthe scaffolds.

5Journal of Nanomaterials

inherent hydrophobic characteristic, which had limited itsfurther application in bone defects treatment [36]. The watercontact angles of the scaffold are shown in Figure 3(f). Asexpected, PHBV scaffold displayed a water contact angle of97:06 ± 1:33°. It was excited to found that the water contactangles of the composite scaffolds significantly decreased withthe increase of ZnO content. For instance, the water contactangle of PHBV/5nZnO composite scaffold sharply decreasedto 58:84 ± 1:29°, which implied that the incorporation ofZnO could achieve a conversion of PHBV scaffold fromhydrophobicity to hydrophilicity. This transformation mightbe attributed to a fact that the ZnO with hydroxyl groups wasfacilitated to the absorption of water molecules and therebyenhancing the hydrophilicity of the composite scaffolds [37].

3.3. Compressive Performance. The compressive performanceof the scaffold plays a critical role in bearing different stressesand offering structural support to the bone tissues [38]. Thecompressive stress-strain curves were measured by mechan-ical testing equipment and are displayed in Figure 4(a). Thecompressive strength and compressive modulus of thecomposite scaffolds calculated from their stress-strain curvesare presented in Figure 4(b). The compressive strength of thecomposite scaffolds firstly raised and then reduced with theincrease of ZnO content. For instance, the compressivestrength of PHBV/3nZnO scaffold increased from 4:1 ± 0:7to 6:4 ± 0:6MPa, which increased by 56% as compared withpure PHBV scaffold. This might mainly be ascribed to thecombination of the increase in the crystallinity of PHBVand a uniform nanoparticle distribution. In detail, theuniformly distributed nanoparticles in the polymer couldaccelerate the orderly stacking of polymer chains, thusenhancing the crystallinity of the composites. The enhancedcrystallinity could efficiently reduce the deformable spaceinside the composites, thereby enhancing their compressivestrength. Meanwhile, the rigid nanoparticles could hinderthe proliferation and development of cracks [39]. Moreover,

the hydrogen bonding interaction between PHBV and ZnOmight absorb a part of energy during the compressive process[40]. However, the compressive strength of the compositescaffold reduced with ZnO content further increasing to5wt.%, which might be ascribed to the aggregation of ZnO.Even so, the scaffold fabricated in this work still fulfilled therequirements for compressive strength of natural cancellousbone, which commonly exhibited a compressive strength of1~10MPa [41]. Furthermore, it could be found that thecompressive modulus of the composite scaffolds improvedwith the continuously increasing of ZnO content. Forexample, the compressive modulus of PHBV/3nZnO scaffoldincreased from 58:6 ± 3:36 to 88:8 ± 5:56MPa, whichincreased by 51.5% in comparison with that of pure PHBVscaffold. This might be attributed to the relatively highmodulus of ZnO nanoparticles, which was in agreement withthe results as reported in the literature [42].

3.4. Antibacterial Properties. Trauma infection was still a bigchallenge in bone repair, which required bone implants tohave antibacterial activity [43, 44]. The bacterial inhibitionrates of the scaffolds with various ZnO contents for 1, 4,and 7 days are shown in Figure 5(a). Obviously, the bacterialinhibition rates of the scaffolds increased with the extensionof days. The bacterial morphologies on the various scaffoldsfor 7 days are shown in Figures 5(b). Obviously, severalrod-like bacteria were adhered and connected to each otheron the surface of PHBV scaffold. On the contrary, thenumber of adhered bacteria was greatly decreased with theintroduction of ZnO. More interestingly, the shapes of bacte-ria became distorted and shriveled, which implied that thecellular structure was damaged. It has been reported thatthe concentration of Zn2+ within 3mg/L shows no cytotoxic-ity to normal cells [45]. In this work, the Zn2+ releases con-centrations of scaffold in the deionized water for 1, 4, and 7days were measured by ICP-OES, and the correspondingresults is displayed in Figure 5(c). All scaffolds showed a slow

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(b)

Figure 4: (a) The compression stress-strain curves, (b) compression strength and compression modulus of composite scaffold. n = 3,∗p < 0:05.

6 Journal of Nanomaterials

and sustained Zn2+ release throughout the whole process.The maximum released Zn2+ concentration of the scaffoldwas 0:3364 ± 0:0024mg/L, which was much lower than3mg/L. Therefore, the scaffolds have no negative effect onthe normal function of cell.

Several antibacterial mechanisms had been proposed tointerpret the antibacterial activity of ZnO nanoparticles, asshown in Figure 5(d). Briefly, it mainly consisted of therelease of Zn2+; the mechanical damage of the cellmembranes resulted from penetration of the nanoparticles,and the generation of reactive oxygen species [46]. In thiswork, the average size of ZnO was 50 nm, which was unlikelyto penetrate into the cell wall to destroy the bacteria [47].Moreover, the production of reactive oxygen species byZnO should be in the irradiation of ultraviolet light [48].Therefore, the release of Zn2+ might be a potential reasonfor the antibacterial activity of the scaffolds. Pasquet et al.

[49] demonstrated that Zn2+ could absorb on the negativelycharged bacterial wall by electrostatic interactions andthereby destroying the normal structure and function of thebacterial membrane, as well as interfering with proteinmetabolism and genetic expression of bacteria.

3.5. Cell Response. The cell response of the scaffolds plays avital in bone repair [50, 51]. Considering the mechanical andantibacterial properties of the scaffolds, the PHBV/3nZnOscaffold was selected to further explore its cell behaviors. Celladhesion was a prerequisite for a series of reactions such asmigration, proliferation, and differentiation [52]. The SEMimages of MG63 cells on PHBV and PHBV/3nZnO scaffoldsfor 1, 3, and 5 days are displayed in Figure 6. Apparently,the spread area of the cell on the scaffolds increased with theprolongation of time. It could be found that the cell on PHBVscaffold presented an ellipse shape in the whole culture

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(d)

Figure 5: (a) Bacterial inhibition rate of E. coli cultured in medium containing various scaffolds for 1, 4, and 7 days; (b) bacterial morphologyon the scaffolds; (c) Zn2+ release of the scaffolds after immersion in deionized water for different time; (d) a schematic for the antibacterialmechanism.

7Journal of Nanomaterials

Day 1 Day 3 Day 5

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/3nZ

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Figure 7: (a) Fluorescence images and (b) optical density of cells on PHBV and PHBV/3nZnO scaffolds after 1, 3, and 5 days of culture. n = 3,∗p < 0:05.

Day 1 Day 3 Day 5

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PHBV

/3nZ

nO20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

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Figure 6: SEM images regarding the cell adhesion behavior on PHBV and PHBV/3nZnO scaffolds for 1, 3, and 5 days of culture.

8 Journal of Nanomaterials

periods, which was attributed to the hydrophobic properties ofthe scaffolds [53]. Compared with the PHBV scaffold, the cellson PHBV/3nZnO displayed a flat and stretched shape after 1day of culture. With the increase of culturing time, moreadhered cells with longer filopodia attachment were observedon the PHBV/3nZnO scaffold, indicating its positive effecton cell adhesion and spreading.

Fluorescence test was performed to further explored theproliferation of cells on PHBV and PHBV/3nZnO scaffoldsand the corresponding fluorescence images for 1, 3, and 5days of cultures are shown in Figure 7(a). Live cells arestained in green, whereas dead cells are stained in red.Clearly, after 1-day culture, the cells on scaffold presented ahealthy and normal polygonal shape, without obvious deadcells, which indicated that these scaffolds provided favorablesurvival environment. Meanwhile, the cell numbers increasedsignificantly after 3 and 5 days of culture. The cell proliferationwas quantitatively described by CCK-8 measurements, andthe results are displayed in Figure 7(b). The optical densityof PHBV and PHBV/3nZnO scaffolds displayed a significantdifference after 5-day culture, implying that the ZnO couldpromote the cell proliferation.

Alkaline phosphatase (ALP) activity, as a typical marker,was widely accepted to reflect the early differentiation ofosteoblasts [54, 55]. The staining images of MG-63 cell onPHBV and PHBV/3nZnO scaffolds after 1, 3, and 5 days ofculture are presented in Figure 8. The ALP-positive areas ofthe cells increased with the increase of culture time. More-over, the MG-63 cells seeded on PHBV/3nZnO scaffoldsdisplayed a more significant ALP staining than that of PHBVscaffolds at the same culture time, which indicated that thePHBV/3nZnO scaffolds could promote the cell differentia-tion. Combining with CCK-8 and live/dead staining assays,it was indicated that PHBV/3nZnO scaffold was moreconducive to cell adhesion, growth, and differentiation. Thereleased Zn2+ could participate in the modulation of cellularsignaling transduction [56]. Meanwhile, the Zn2+ could regu-late the interaction between signal peptides and extramem-brane receptors, thereby improving the cell behaviors [57].In addition, Zn is an important trace element, which widelyinvolves in synthesis of several nucleic acid and protein [58].

4. Conclusion

In this study, ZnO nanoparticles were incorporated intoPHBV scaffolds fabricated by the SLS technique, aiming atimproving their mechanical properties and antibacterial activ-ities. The results indicated that a scaffold with 3wt.% ZnOexhibited a uniform dispersion and simultaneously could pro-vide a nucleating site for the orderly stacking of PHBV chains.The relative crystallinity of PHBV/3nZnO scaffold remarkablyincreased from 38% to 64% in comparison with that of purePHBV scaffold. The improved crystallinity could effectivelyenhance the compression strength andmodulus of the scaffoldby 56% and 51.5%, respectively. Moreover, Zn2+ released bythe scaffolds could efficiently inhibit the growth of E. coliand promote the cell behaviors in terms of cell proliferationand differentiation. All these positive results confirmed thatthe scaffold was one potential bone scaffold material.

Data Availability

The case data used to support the findings of this study areincluded within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was supported by the following funds: (1) TheNatural Science Foundation of China (51935014, 51905553,81871494, 81871498, and 51705540); (2) Hunan ProvincialNatural Science Foundation of China (2019JJ50774, 2018JJ3671, and 2019JJ50588); (3) The Provincial Key R&DProjects of Jiangxi (20201BBE51012); (4) JiangXi ProvincialNatural Science Foundation of China (20192ACB20005); (5)Guangdong Province Higher Vocational Colleges & SchoolsPearl River Scholar Funded Scheme (2018); (6) The Projectof Hunan Provincial Science and Technology Plan (2017RS3008), (7) Shenzhen Science and Technology Plan Project(JCYJ20170817112445033), (8) Innovation Team Project on

200 𝜇m 200 𝜇m 200 𝜇m

200 𝜇m 200 𝜇m 200 𝜇m

Day 1 Day 3 Day 5

PHBV

PHBV

/3nZ

nO

Figure 8: ALP staining images of MG-63 cells on PHBV and PHBV/3nZnO scaffolds after 1, 3, and 5 days of culture.

9Journal of Nanomaterials

University of Guangdong Province (2018GKCXTD001), and(9) Technology Innovation Platform Project of ShenzhenInstitute of Information Technology 2020 (PT2020E002).

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